Drug discovery remains a top priority in medical science. The phenomenon of drug resistance has heightened the need for both new classes of pharmaceutical as well as novel modes of action. In recent years we have worked to develop a distinct approach to drug design that involves both recognition and subsequent irreversible inactivation of therapeutic targets. The basic drug design strategy incorporates a protein recognition domain and a metal binding domain, where the latter mediates irreversible inactivation of the therapeutic target. Inactivation is both catalytic and multiturnover, while the incorporation of both binding and catalytic centers provides a double-filter mechanism for improved target selectivity and lower dosing. The specific aims for the proposed funding period will focus on improving on lead metallopeptides of potential therapeutic value against cardiovascular targets, the demonstration of efficacy in animal models, and elaborating the mechanism of action of such metallodrugs against protein targets. Our overall goals can be summarized as follows. (1) Design and evaluate triple-action metallodrugs that target three key cardiovascular enzymes (ACE, ECE-1 and NEP). (2) Optimization of in vivo stability and efficacy through tuning of peptide sequence and amino acid configuration. (3) Evaluate the mechanism of catalytic inactivation of protein targets. Optimization of the intrinsic reactivity of the catalytic metallodrugs toward metal-mediated degradation of target molecules will require an advanced understanding of the mechanism of catalytic cleavage (to be achieved by use of a variety of kinetic and mass spectrometric methods). These studies will combine fluorogenic activity assays of metallopeptide inactivation of ACE, ECE-1 and NEP, as well as animal studies of a spontaneously hypertensive rat model. Mass spectrometric analysis of modified proteins and proteolytic digests will reveal details of side-chain modification that underlie catalytic inactivation. The impact of substituting L- for D-amino acids on metallodrug activity, target recognition, and in vivo stability will be investigated. The animal studies will also serve to validate initial observations that delivery of the metal-free peptide alone is sufficient for activity, with recruitment of metal cofactor from the cellular environment following uptake. PUBLIC HEALTH RELEVANCE: Drug discovery remains a top priority in medical science. The phenomenon of drug resistance has heightened the need for both new classes of pharmaceutical as well as novel modes of action. In recent years we have worked to develop a distinct approach to drug design that involves both recognition and subsequent irreversible inactivation of therapeutic targets. This concept allows for improved target selectivity and lower dosage requirements and will be further developed against cardiovascular protein targets in both solution studies and animal models.